109 Isolation and characterization of myometrium and decidual cell-derived extracellular vesicles

Abstract

Extracellular vesicles (EVs-exosomes; 30–160 nm) contribute to intercellular communication by transporting various signaling molecules. The cargo in these EVs may reflect the physiological or pathophysiological state of the source cells. Prior reports have shown that fetal tissue derived EVs can signal parturition by inducing functional changes in maternal uterine tissues; however, little is known about maternal cell derived EVs and their role in pregnancy. This study isolated and characterized EVs derived from decidual and myometrial cells under normal and inflammatory/oxidative stress (OS) conditions and determined their cargo contents. Decidual and myometrial cells were grown under standard conditions (control) or exposed to cigarette smoke extract (CSE) or tumor necrosis factor-α (TNF-α) as proxies for OS and inflammation. EVs were isolated from media using differential ultracentrifugation. The isolated EVs were quantified (ZetaView), characterized for size and morphology (cryo electron microscopy), and tested for markers (Dot Blot). Proteome cargo analysis was done with mass spectroscopy followed by bioinformatics analysis using ingenuity pathway analysis. EVs from both decidua and myometrial cells were round (Fig 1A) and expressed EV specific tetraspanins and ESCRT protein markers (Fig 1B). Size and quantity of isolated EVs are represented in figure 1C. Decidual EVs contained 1419 common proteins between all 3 groups. There were 10 unique proteins in control, 15 in CSE and 24 in TNF-α. Myometrial EVs contained 1288 common proteins between all 3 groups. There were 32 unique proteins in control, 39 in CSE and 14 in TNF-α (Fig2A). Both CSE and TNF-α upregulated various proteins isolated from EVs from both cell lines (Fig 2B). This is the first study to isolate and characterize maternal uterine cell derived EVs in pregnancy. OS and inflammation packages different protein cargo in EVs from both cell types and they are likely to have specific functions in recipient cells. The impact of maternal EVs on fetal tissues are being investigated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)